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Repair mechanisms

Repair mechanisms. 1. Reversal of damage 2. Excision repair 3. Mismatch repair 4. Recombination repair 5. Error-prone repair 6. Restriction-modification systems. 1. Reversal of damage. Enzymatically un-do the damage a) Photoreactivation b) Removal of methyl groups. 5. 5. 6. 6.

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Repair mechanisms

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  1. Repair mechanisms 1. Reversal of damage 2. Excision repair 3. Mismatch repair 4. Recombination repair 5. Error-prone repair 6. Restriction-modification systems

  2. 1. Reversal of damage • Enzymatically un-do the damage • a) Photoreactivation • b) Removal of methyl groups

  3. 5 5 6 6 d-ribose d-ribose Photolyase breaks apart pyrimidine dimers Photolyase breaks the bonds between the dTs

  4. 1a. Photoreactivation • Photolyase: binds a pyrimidine dimers and catalyzes a photochemical reaction • Breaks the cyclobutane ring and reforms two adjacent T’s • 2 subunits, encoded by phrA and phrB.

  5. 2. Excision repair • General Process: • remove damage (base or DNA backbone) • ss nick/gap provides 3’OH for DNA Pol I initiation • DNA ligase seals nick • Nucleotide excision repair: • Cut out a segment of DNA around a damaged base. • Base excision repair: • Cut out the base, then cut next to the apurinic/apyrimidinic site, and let DNA Pol I repair

  6. Discovery of mutants defective in DNA repair uvr-

  7. polA mutants are defective in repair wt polA mutant DNA synthesis in vitro Survival after UV in vivo

  8. UvrABC excision repair

  9. Cleavage and helicase

  10. Fill in with polymerase and ligate

  11. Mutations in excision repair in eukaryotes can cause xeroderma pigmentosum (XP) HumanAnalogous GeneProtein Function to E. coli: XPA Binds damaged DNA UvrA/UvrB XPB Helicase, Component of TFIIH UvrD XPC DNA damage sensor XPD Helicase, Component of TFIIH UvrD XPE Binds damaged DNA UvrA/UvrB XPF Works with ERRCI to cut DNA UvrB/UvrC XPG Cuts DNA UvrB/UvrC

  12. 2b. Base excision repair Incorrect or

  13. Excision and filling in by DNA PolI

  14. 3. Mismatch repair • Action of DNA polymerase III (including proofreading exonuclease) results in 1 misincorporation per 108 bases synthesized. • Mismatch repair reduces this rate to 1 change in every 1010 or 1011 bases. • Recognize mispaired bases in DNA, e.g. G-T or A-C base pairs • These do not cause large distortions in the helix: the mismatch repair system apparently reads the sequence of bases in the DNA.

  15. Role of methylation in discriminating parental and progeny strands • dam methylase acts on the A of GATC (note that this sequence is symmetical or pseudopalindromic). • Methylation is delayed for several minutes after replication. • Mismatch repair works on the un-methylated strand (which is newly replicated) so that replication errors are removed preferentially.

  16. Action of MutS, MutL, MutH • MutS: recognizes the mismatch (heteroduplex) • MutL: a dimer; in presence of ATP, binds to MutS-heteroduplex complex to activate MutH • MutH: endonuclease that cleaves 5' to the G in an unmethylated GATC, leaves a nick

  17. MutH, L, S action in mismatch repair#1

  18. MutH, L, S action in mismatch repair #2

  19. Mismatch repair: Excision of the misincorporated nucleotide

  20. Eukaryotic homologs in mismatch repair • Human homologs to mutL (hMLH1) and mutS (hMSH2, hMSH1) have been discovered, because ... • Mutations in them can cause one of the most common hereditary cancers, hereditary nonpolyposis colon cancer (HNPCC).

  21. 4. Recombination repair: retrieval of information from a homologous chromosome

  22. 5. Error-prone repair • Last resort for DNA repair, e.g when repair has not occurred prior to replication. How does the polymerase copy across a non-pairing, mutated base, or an apyrimidinic/apurinic site? • DNA polymerase III usually dissociates at a nick or a lesion. • But replication can occur past these lesions, especially during the SOSreponse ("Save Our Ship"). • This translesionsynthesis incorporates random nucleotides, so they are almost always mutations (3/4 times)

  23. Role of umuC and umuD genes in error-prone repair • Named for the UV nonmutable phenotype of mutants with defects in these genes. • Needed for bypass synthesis; mechanism is under investigation. E.g. these proteins may reduce the template requirement for the polymerase. • UmuD protein is proteolytically activated by LexA.

  24. UV damage DNA replication Translesional synthesis (error-prone) DNA Pol III UmuD 2 UV damage, increase RecA:ssDNA UmuD’ 2 UmuC UmuC Activate protease Induce umuC+, umuD+ Pol III alpha epsilon beta Polymerase for translesional synthesis DNA damage checkpoint control UmuC, UmuD in error-prone repair Graham Walker

  25. SOS response is controlled by LexA and RecA

  26. LexA, RecA in the SOS response

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